This invention relates to the use of procollagen (III) propeptides and related substances for treating fibrotic diseases and a method for producing and renaturing recombinant N- and/or C-terminal procollagen (III) propeptides. Said procollagen (III) propeptides and related substances are suitable for treating fibrosis of any type, of any organ manifestation. The invention also relates to a method for producing renatured N-terminal procollagen (III) propeptide and/or C-terminal procollagen (III) propeptide.
Collagen Biosynthesis.
The collagens of types I and III are synthesized as prepropeptides and are extensively modified posttranslationally. Among the intracellular modifications are glycosylations, enzymatic hydroxylation reactions involving lysine and proline in its 3- and 4-positions. The modified propeptides spontaneously assemble into [a1 (III)]3 homotrimers in the case of collagen (III). In the case of collagen type I mostly [a1(I)]2a2(I) heterotrimers as well as—to a lesser extent—[a1(I)]3 homotrimers are formed. After exocytosis, the propeptides are first cleaved at the C-terminus of the nascent collagen and then at the N-terminus by a set of specific endoproteases. The cleavage resulting in the C-terminal procollagen propeptide (PIIICP) is catalyzed by the procollagen C-proteinase that hich is identical to the Bone Morphogenetic Protein-1. Different tissue-specific expression patterns of different splice variants of the BMP-1 protein have been discovered.
The N-terminal procollagen propeptide of collagen I (PINP) is cleaved off by the same N-proteinase that also digests the N-terminal procollagen propeptide of collagen type II. By contrast, N-terminal procollagen (III) propeptide (PIIINP) is cleaved by off by a proteinase activity distinct from the N-proteinase (I and II). The responsible enzyme is called procollagen N-proteinase type III.
PIIICP.
PIIICP occurs as a trimer consisting of three identical monomeric PIIICP subunits that are linked by intermolecular disulfide bridges. Theoretical structural considerations and site-directed mutagenesis experiments with so-called collagen mini genes have led to the conclusion that at least 4 and probably 6 cysteine residues of each monomeric PIIICP subunit are involved in intramolecular disulfide bridge formation. It is likely that only the cysteine residues in positions 51 and 68 are involved in intermolecular disulfide bridge formation. It has been observed, however, that the region around these cysteine residues is critical for the correct formation of intramolecular disulfide bridges because a Cys→Ser mutation in that region leads to impaired intramolecular disulfide bridge formation. On the other hand it has been observed that the trimerization of collagen (III) fibrils proceeds even when interchain cystine bridge formation has become impossible by a Cys→Ser mutation in position 51.
So far, there have been no reports about the quantification of PIIICP except in the patents: An immunoassay for procollagen-III-C-terminal propeptide (WO09924835A2 and EP00988964A1).
There are no reports about pharmacokinetic data for PIIICP. For PICP, clearance and mode of elimination have been investigated. For 125I-labeled PICP, an elimination from the serum by the mannose-6-phosphate receptor has been reported. PIIICP could also be cleared from the circulation by this receptor as PIIICP may also be glycosylated at position Asn173. This mechanism can safely be excluded for recombinant PIIICP from E. coli, however, as the protein is not glycosylated when expressed in this host.
With regard to the physiological role of PIIICP, there is no information available in the literature. With regard to the biological effects of the C-terminal propeptide of collagen type I, different effects have been described in the literature.
The nucleotide sequence of human PIIICP has been deposited in the Genebank (Accession No. X14420 and X01742). The amino acid sequence of this peptide is shown in FIG. 1(I) as an example. The propeptide sequence is indicated in the appendix in the context of the whole procollagen sequence C-terminal of the procollagen C proteinase cleavage site.
In fibroblast cell culture, a reduction of collagen production by 80% was measured when intact PICP was present at a concentration of 40 nM, while it was decreased by 30% at 10 nM. However, these changes in the protein biosynthesis very well correlated with the measured changes at the level of transcription. This lead to the speculation that PICP exerts a regulatory effect at the level of transcription.
For intact rat PICP, isolated from fibroblasts, an inhibiton of collagen biosynthesis was also demonstrated with cell cultures of hepatic stellate cells. At a concentration of 33.3 nM an inhibitory effect of 66% was measured that increased to 83% at a concentration of 133.2 nM, respectively, Changes in mRNA concentrations affected by PICP were not investigated. It was shown in this series of experiments, however, that the inhibitory effect was strongly dependent on the structure of the protein. A covalent modification of PICP induced by the exposure to acetaldehyde lead to a marked reduction of the effect on collagen biosynthesis.
The effects of overlapping synthetic polypeptides derived from the PICP sequence in a fibroblast cell culture model are reported ambiguously in the literature.
An inhibitory effect at the level of the rate of biosynthesis was observed with a polypeptide consisting of 22 amino acid residues (residues 225 to 246).
By contrast, a further polypeptide consisting of 45 amino acid residues (residues 197 to 242) lead to an increased rate of biosynthesis for collagens of types I and III as well as for fibronectin—contrary to all previous results. At a concentration of 45 μM an increase of collagen biosynthesis by a factor of 3.3 and of fibronectin biosynthesis by a factor of 6.1 was observed in human lung fibroblasts after 4 h. After 8 a maximal stimulation of collagen biosynthesis by a factor of 6- to 8-fold was measured.
However, the stimulatory effect was dependent on the degree of confluence of the cells. While an effect was observed in subconfluent fibroblast cells, this effect could not be demonstrated in confluent cells. The effects were neither cell type- nor species-specific. In further experiments the polypeptide sequence sufficient for eliciting the stimulatory effect could be reduced to a pentapeptide (amino acid residues 212 to 216). In these experiments, 80% of the maximal stimulation was observed. The effect was notably more pronounced with fibronectin (5- to 11-fold increase) in comparison with collagen type I (4- to 7-fold increase). In parallel to the experiments focused on the protein level, the mRNA concentrations of the concerned genes were investigated. For collagen as well as for fibronectin, no concentration changes were measured at the mRNA level. According to these data, the synthetic polypeptides exerted their effects on the stimulation of collagen biosynthesis at the posttranscriptional level.
In vivo experiments with PIIICP or related substances have not been reported in the literature so far.
PIIINP
PIIINP occurs as a trimer consisting of three identical monomeric PIIINP subunits that are linked by intermolecular disulfide bridges. The PIIINP molecule is structurally divided into three domains. The most N-terminally located domain (Col1) consists of a globular structure and contains several intramolecular cystine bridges. The C-terminally adjacent Col3 domain possesses a collagen-like structure characterized by periodic Gly and Pro residues. This domain assembles into a characteristic triple-helical collagen-like structure. The Col2 domain encompasses those parts of the procollagen telopeptide region that are N-terminal of the N-proteinase cleavage site. The monomeric PIIINP strands are assembled parallel to each other in this region.
Characteristically, the Col2 domain contains two cysteine residues that are both involved in intermolecular disulfide bridge formation and that are of eminent importance for the trimeric structure of PIIINP. An oligosaccharide glycosylation site is located in the vicinity of the N-proteinase (III) cleavage site. Eight amino acid residues C-terminal of the propeptide cleavage site, one of the four lysyl residues is located which are oxidatively desaminated into aldehydes before the collagen fibrils become covalently crosslinked.
A special characteristic of collagen type III is that a fraction of the N-terminal propeptides is not cleaved off the procollagen trimer. This so-called pN-collagen type III is still incorporated into fibrils. The form of these fibrils is described in the literature in different ways: as thin fibrils that are associated with collagen type I or as pearl necklace-like fibrils that associate to become net-like structures. Electron-microscopically, pN-collagen of type III has the appearance of a “barbed wire”. The presence of pN-collagen type III on the surface of collagen fibrils could play a role in the regulation of the diameter of the fibrils.
With regard to the stability of PIIINP in the body, half lives between 2 min and 239 min have been reported. The determined values varied considerably depending on the model and/or the labelling of the antigen. In rats a clearance of the N-terminal propeptide of collagens III and I from the serum—independently of the antigen species—by the scavenger receptor on liver endothelial cells has been reported (Melkko 1994). Endocytosis was mediated by the same receptor for both proteins.
The amino acid sequence of human PIIINP is deposited in the Genebank database with the accession number X14420. As an example, the amino acid sequences of this propeptide are shown in FIG. 1(II). The propeptide sequence is indicated in the context of the whole procollagen sequence N-terminal of the procollagen N proteinase type III cleavage site.
PIIINP has so far mostly been described as a marker of fibrosis. It can be used in the context of possible therapies of liver fibrosis as a possible non-invasive parameter to follow the course of the disease.
For PIIINP, research about its role as a feedback inhibitor of collagen biosynthesis in cell culture systems and in cell-free lysates has been published. For the N-terminal propeptides of collagens type I and type III as well as for the Col 1 domain of PINP, a concentration-dependent cell-specific inhibition of collagen production of the a1(I) and a2(I) chains has been measured in the fibroblast cell culture system. Protein concentrations from 0.5 μM to 6 μM were used. The inhibition was in the range between 30% to 50% in comparison with control experiments. In these experiments experimental evidence was provided that the rate of translation was influenced by the propeptides and by their fragments. These data were supported by the localization of the internalized proteins in the vicinity of the endoplasmatic reticulum.
In addition, experiments with the Col 1 domains of both N-terminal propeptides were carried out in the cell-free reticulocyte system. Protein concentrations ranging from 0.4 to 3.2 μM were used. A concentration-dependent inhibition of collagen (I) synthesis was measured. In both cases, an inhibition between 38% and 71% in comparison with control experiments was measured. When very high protein concentrations (8 μM) were used, it was demonstrated that the inhibitory effect on collagen translation could not be further increased.
When the mechanism of action of PINP was investigated a system for the recombinant cyotosolic expression of PINP in fibroblast cell culture was also examined. While the measured collagen (I) mRNA concentration was unchanged, the rate of biosynthesis of the corresponding protein was reduced. It was therefore regulated at the post transcriptional level.
These results for PINP are supported by experiments with skin fibroblasts from dermatosparactic sheep. In the homologous human disease, Ehlers Danlos syndrome of type VIIa or VIIb, a mutation within the N-proteinase cleavage site of procollagen type I occurs. Consequently, PINP cannot be cleaved off. In cell culture, these cells which lack the PINP feedback mechanism in comparison with heterozygous control fibroblasts, displayed a proportion of 15 to 20% of collagen biosynthesis compared to the total cellular biosynthesis (control fibroblasts 2 to 4%).
Recombinant Production of Procollagen III Propeptides.
The recombinant production of procollagen (III) propeptides has been reported in a number of publications. The recombinant production of a collagen a2(I) mutant in so-called A2 cells derived from the rat liver epithelial cell line W8 which is in turn deficient for collagen a2(I) is described. The recombinant expression of collagen a1(III) minigenes has been described more recently.
Recently, the production of PIIICP in Zf9 cells as a trimeric protein has been described. The recombinant protein could only be produced in small quantities for analytical purposes, however. The recombinant production of PIIICP in E. coli was described in the patent applications: An immunoassay for procollagen-III-C-terminal propeptide (WO09924835A2 and EP00988964A1) and in Burchardt, 1998. The yields were above 20 mg/l fluid culture medium with this expression method. The majority of the recombinant protein was in the form of inclusion body protein, however, and had to be purified using denaturing methods of dissolution. The expressed proteins also contained an N-terminal His tag, so that they could be purified in denaturing solvents over a Ni-NTA column. For chronic in vivo applications, these proteins were less suitable because of the potential immunogenicity of the His tag and because the biological half-life of the recombinant proteins may be decreased for this reason. They occurred mostly in a monomeric form when the methods disclosed in this application were used. Their solubility was too low for most therapeutic applications. When a concentration of approximately 10 μg/ml was exceeded, the recombinant PIIICP precipitated from aqueous solutions.
The recombinant expression of human PIIINP in E. coli has been described in the patent application: Monoclonal antibody and assay for detecting PIIINP (WO09961477A2), and the expression of murine PIIINP has been reported in Kauschke, 1999. The yields were at approximately 5 mg/l fluid culture medium with this expression method. These expressed proteins also contained an N-terminal His tag , so that they could be purified in denaturing solvents over a Ni-NTA column. For chronic in vivo applications, these proteins were also less suitable because of the potential immunogenicity of the His tag and because the biological half-life of the recombinant proteins may be decreased for this reason. The solubility of PIIINP in aqueous solutions was too low for most therapeutic applications.
Fibrotic Diseases.
Fibrotic diseases are defined as a diverse group of diseases that are associated with a qualitatively altered collagen production or with an increased deposition of collagen in the exrtracellular space. To this group of diseases belong, among others, systemic or localized scleroderma, liver fibrosis of various etiologies, alcoholic cirrhosis, e.g. alcoholic liver cirrhosis, biliary cirrhosis, hepatitis of viral or other origin, veno-occlusive disease, idiopathic interstitial fibrosis, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, acute pulmonary fibrosis, acute respiratory distress syndrome, perimuscular fibrosis, pericentral fibrosis, dermatofibroma, kidney fibrosis, diabetic nephropathy, glomerulonephritis, keloids, hypertrophic scars, joint adhesions, arthrosis, myelofibrosis, corneal scaring, cystic fibrosis, muscular fibrosis, Duchenne's muscular dystrophy, esophageal stricture, retroabdominal scaring, Crohn's disease, ulcerative colitis, atherosclerotic alterations, pulmonary hypertension, angiopathy of the arteries and veins, aneurysms of large vessels.
Further fibrotic diseases are induced or initiated by scar revisions, plastic surgeries, glaucoma, cataract fibrosis, corneal scaring, graft vs. host disease, tendon surgery, nerve entrapment, Dupuytren's contracture, OB/GYN adhesions, pelvic adhesions, infertility, peridural fibrosis, diseases of the thyroid gland or the parathyroids, metastatic bone disease, multiple myeloma, or restenoses. In many of the aforementioned diseases, a successful therapy has not been established so far. In others, a need for improved approaches or for the reduction of undesired side effects exists.
From the aforementioned it follows that there is a further need to supply efficacious drugs against fibrotic diseases. The solution to this task is achieved by the embodiments presented in the examples.